The experiments in this proposal are designed to establish and characterize a tissue culture model for the study of reactive astrogliosis. Removal of the retina by surgical enucleation severs the axons of retinal ganglion cells which comprise the optic nerve. The procedure causes the axons to degenerate and ultimately leads to the formation of a glial scar in vivo. When the central neurons system (CNS) is injured, reactive astrocytes play a role in "repair" of degenerating nervous tissue by removing cell debris and reconstituting the glial limiting membrane at the site of trauma. A considerable number of questions on the subject of reactive astrocytes and their role in sprouting and regeneration in the CNS remain unanswered. This proposal describes the development of an in vitro model which can be used for a systematic examination of the causes, characteristics and consequences of the reaction of astrocytes to CNS injury. Experiments in the first specific aim will quantitatively document the time course of optic nerve degeneration and reactive astrogliosis in vivo. These experiments will utilize electron microscopy and immunocytochemistry to establish a list of criteria which can be used to identify reactive astroglia in vivo. The purpose of the next set of experiments is to determine if reactive astroglia in explants of glial scars will remain reactive in vitro (2). To determine if reactive astrogliosis can occur in vitro, a comparison between optic nerve degeneration and glial scar formation in vivo and in vitro will be made (3). Preliminary results indicate that reactive astrogliosis does occur in culture but the sequence of optic nerve degeneration appears to be altered. Factors contributing to the differences between optic nerve degeneration in vivo and in vitro will be explored (4). Dissociated cell cultures of glial scars will also be prepared. Studies of astrocytes dissociated from glial scars will provide insight into the influence of intercellular interactions on the expression of reactive astrogliosis. The question of whether the reaction of astrocytes to injury is a single-step or a multi-step process will also be addressed using dissociated cell cultures of glial scars. Having established this culture model, it will be used to examine if factors implicated as causes for astroglial reactivity in vivo can induce reactive astrogliosis in vitro (5). These factors include: products of neuronal degeneration and myelin basic protein and products of myelin degeneration.